Growth plate development is a critical step in endochondral bone formation. Delayed cell differentiation in the growth plate can produce chondrodysplasia, hypophosphatasia, rickets, and growth retardation. The orderly progression of chondrocyte differentiation is maintained by hormones, nutritional components, and autocrine and paracrine factors produced by chondrocytes. The concentration of Ca2+ in the extracellular fluid ([Ca2+]e) plays a key role in growth plate cartilage development. Certain forms of childhood rickets are due to extremely low Ca2+ intakes. The bones and cartilage in rickets are soft and deformed and growth is stunted. We found that raising the [Ca2+]e directly promotes the terminal differentiation and mineralization of cultured mouse growth plate chondrocytes (mGPCs). Like Ca2+, insulin-like growth factor-1 (IGF1) promotes GPC differentiation. High Ca2+ also increases IGF1/IGF1R signaling capacity in mGPCs, suggesting that IGF1R-mediated signaling may be involved in the high [Ca2+]e-induces cell differentiation. The signaling mechanisms responsible for the effects of Ca2+ in the growth plate have not been defined. The Ca2+-sensing receptor (CaR) is strongly expressed in mGPCs. Altering CaR expression and function profoundly impacts the differentiation of these cells. To determine whether the CaR is responsible for extracellular Ca2+-sensing by cartilage in vivo, we generated mice [CartCaR(-/-)] with tissue-specific knockout of the CaR in chondrocytes by a Cre-lox recombination technique. These mice die in gestation with malformed skeletons. We also developed a tamoxifen-inducible, chondrocyte-specific CaR knockout mouse [(Tam-CartCaR(-/-)]. These mice grow poorly during their postnatal life and have abnormally expanded growth plates with delayed mineralization in their hypertrophic zones -- features seen in rickets. These data strongly support a role for the CaR in growth plate development. We will use these models to address the hypothesis that a high [Ca2+]e by activating CaRs stimulates ERK1/2 and PLC activity, increases the intracellular [Ca2+], enhances the expression of and signaling by IGF1 and the IGF1R, and promotes the terminal differentiation of GPCs. We propose the following AIMs: (1) to determine the role of the CaR at specific stages of growth plate development by inducing CaR gene deletion with Tam at timed intervals before and after birth; (2) to determine whether a high [Ca2+]e alters chondrocyte function by regulating the expression of and signaling by IGF1 and the IGF1R in chondrocytes; (3) to assess the contribution of IGF1 and IGF1R-mediated signaling to the responses of GPCs to changes in the [Ca2+]e; and (4) to identify the high [Ca2+]e-induced signaling responses that are mediated by the CaR and are responsible for changes in the growth, survival, and differentiation of chondrocytes. Successful completion of these studies will provide insights into how the CaR interacts with local IGF1/IGF1R signaling pathway to modulate GPC differentiation and growth plate development. Our studies will advance the field of CaR biology and may provide a potential pharmaceutical target for disorders of growth plate function in childhood. PUBLIC HEALTH RELEVANCE: Growth plate cartilage, which is located at the ends of the bones, allows them to grow bigger. Chondrocytes from growth plate cartilage are capable of sensing changes in the concentrations of Ca2+ in their surroundings and adjusting their activities accordingly. Changes in the concentrations of Ca2+ near growth plate chondrocytes influence their whole program of gene expression, matrix protein synthesis, and mineral deposition. This proposal will investigate how Ca2+ acts on these chondrocytes and will help us understand how to treat disorders of growth and poor mineralization like rickets.